Download - INTRODUCTION BIOCHEMISTRY · I. INTRODUCTION Inthe metabolism of fresh water or terrestrial bacteria andfungi, thehalogensarenotgenerally considered functional elements. Despite their

AN INTRODUCTION TO THE ORIGIN AND BIOCHEMISTRY OFMICROBIAL HALOMETABOLITES

MILTON A. PETTYLederle Laboratories, American Cyanamid Company, Pearl River, New York

I. Introduction ........................................... 111A. Incidence and Origin .............................................. 111

II. Relationships of Halometabolites ................................ 112A. Chemical.............................................. 112B. Biological .............................................. 113

III. Factors Influencing Biosynthesis of Halometabolites ....................... 118A. Genetic.............................................. 118B. Chemical.............................................. 120C. Inhibition of Halogenation ............................................... 121

IV. Halogen Interchangeability .............................................. 122V. Biosynthesis and Mechanism of Halogenation........................................... 122VI. Function of Halometabolites .............................................. 125

VII. References ............................... 125

I. INTRODUCTIONIn the metabolism of fresh water or terrestrial

bacteria and fungi, the halogens are not generallyconsidered functional elements. Despite theirestimated abundance, wherein chlorine is rankedas the eleventh most common element, and therelative aqueous solubility of halide salts, little isknown of the metabolic processes involving theseelements especially in the lower plants. Only in re-cent time has there been interest in halogen-con-taining metabolites and their biogenesis. The onlywestern review on halometabolites is by Bracken(13) who discussed 12 of the presently known 29"natural" chlorometabolites. It is the purpose ofthis review to consider the microbiology of theproducers and the biosynthesis of halometabolitesand their analogues.1 As used here, the termhalometabolite defines an organic halogen-con-taining molecule synthesized by a living organ-ism from the metabolism of ionic halide.

Historically, the first record of a microbialhalometabolite is given by Zopf (120) who iso-lated diploicin (Table 1: 9a) from a lichen. Adeshalo-analogue of diploicin has yet to be re-ported. The first record of a deshalo-analogue,atranorin (Table 1: 8b), of a subsequently dis-covered halometabolite, chloroatranorin (Table1: 8a), is credited to Paterno and Oglialoro in1877 (22). This history presents an anomaly,since the more common sequence of disclosure is

1 Literature published prior to February 1960forms the basis for this review.

for halometabolite discovery to precede that ofdeshalo-analogue. Of the halometabolites known,bromine derivatives are not produced with thesame ease or abundance as their chloro-analogues.In fact, the brominating microorganisms producemore deshalo-analogue than bromometabolite inthe presence of bromide. Since conventionalnutrient media commonly contain chloride eitheras a result of using natural materials or throughthe addition of an inorganic chloride, e.g.,Czapek-Dox solution, opportunity for biosyn-thesis of chlorometabolites is commonly afforded.Even media of supposedly defined nature, suchas Raulin-Thom which has no deliberately addedchloride, have afforded sufficient chloride as animpurity to result in the biosynthesis of a halo-metabolite (89). Iodo- or fluorometabolites havenot been reported despite attempts by several in-vestigators to obtain them from microorganismsnormally producing chlorometabolites.

A. Incidence and OriginOne is led to speculate on the relative inci-

dence and origin of halometabolites. In the caseof a relatively common and simple nonhalo-organic metabolite, synthesized by a number ofmicroorganisms and perhaps in several analogousforms, should one expect to find this to be pro-duced as a halo-analogue by a few species? Theexample of atranorin and chloratranorin, and thedepsidones (Table 1: 9) is cited. If this be true,and since usnic acid:

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MILTON A. PETTY

0

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has been reported as the most widely distributedacid in lichens, occurring in more than 90 differ-ent species, then one might expect to find chlo-rousnic acid in a few species of lichens. This hasnot been done. With the more complex but struc-turally related metabolites produced by only afew species, should one expect to find a culturecapable of producing a halo-analogue? The factsdo not seem to support this approach. For exam-ple, a number of strains of Penicillium urticae(Penicillium griseofulvum) produce griseofulvin(Table 1: lOd) along with a smaller quantity ofthe deshalo-analogue (65), and only some of thegriseofulvin-producing strains of Penicilliumnigricans made small amounts of dechlorogriseo-fulvin (Table 1: lOe). With Streptomyces aureo-faciens, rarely does a natural isolant producemeasurable amounts of tetracycline (Table 1:12h) in the presence of chloride, since the normaltendency is to produce chlortetracycline (Table1: 12e). There is no authenticated report of theisolation from nature of an S. aureofaciens strainincapable of producing chlortetracycline but pro-ducing instead "pure" tetracycline. All culturescapable of producing essentially "pure" tetra-cycline in the presence of available chloride areknown to be laboratory-derived mutants andnot isolants from nature. The halogenating mech-anisms are therefore dominant in the wild formsof halometabolite producers.

Experiences with S. aureofaciens and Strepto-myces venezuelae do not indicate that they areubiquitous. Neither of these species was found inmore than 0.03 per cent of soil samples fromvarious countries (E. J. Backus, personal com-munication). Furthermore, out of 2475 culturesof Streptomyces species isolated from Korean soils,only 6 were found to produce a chloramphenicol-like activity (20). These facts point to either arelative rarity, or an area of scant knowledgeconcerning the incidence of most halometaboliteproducers in nature. Producers of correspondingdeshalo-analogues seem to be even rarer, exceptfor the one lichen metabolite, atranorin.

II. RELATIONSHIPS OF HALOMETABOLITES

A. Chemical

Table 1 presents a total of 37 halometabolitesof natural or laboratory origin, represented by12 different structural groups in figure 1. Thestructure of 10 of the metabolites is yet to bedetermined. Excluded from this group are thepenicillins which have been biosynthesizedthrough the addition of chloro-precursors. Pro-posed structural configurations of the halo-metabolites are presented in figure 1. Of the 37halometabolites, 29 are of natural or conventionalculture origin. All of the 29 "natural" halo-metabolites are chlorine derivatives. Bromo-derivatives of three of the "natural" chloro-metabolites have been biosynthesized and afourth, bromo-demethyltetracycline (Table 1:12a) is described in patent literature. Of the 29"natural" chlorometabolites, 7 have dechloro-analogues of biosynthetic origin (Table 1: 4b, 4c,8b, 9e, 9f, i0e, lib, 12f, 12h).

There may be some merit in considering a dis-tinction between chemically and biologically syn-thesized analogues. For example, neither rotiorin(VII) nor N-propionyl-p-nitrophenylserinol (III;R'==CH3, R9--H) are the respective chemicaldeshalo-analogues of selerotiorin (VI; R=Cl)and chloramphenicol (III; R'-=R2=Cl), butthey could well be considered bio-analogues.Of the 29 "natural" halometabolites, with the

exception of chloramphenicol, all have the halo-gen attached to a cyclic carbon of a 5- or 6-mem-bered ring. However, the acetylserinol moiety ofchloramphenicol might be an intermediate to themethyl-pyrrole ring type of moiety of pyoluteorin(IV) since a dehydration reaction could close typeIII structure into a type IV structure. Onlycaldariomycin (I) and chloramphenicol (Table 1:4a) have two chlorines attached to one carbon.It is not clear where the chlorines are attached inpyoluteorin (IV). The chlorine to carbon ratio isthe greatest in drosophilin A (II), and it seemsscarcely possible that this compound, p-methoxy-tetrachlorophenol, is a product of a fungus sincethe polychlorophenols are potent fungicides.Somewhat along the same line, mollisin (V) isclosely related chemically to the fungicidedichlone, 2,3-dichloro-1,4-naphthoquinone. Thestructural homology of the depsides, depsidones,and spirans is interesting, with geodoxin (XIII)bridging the gap between the latter two groups.

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MICROBIAL HALOMETABOLITES

One substance, pannarin (XI), appears to haveescaped the attention of the better known ab-stracting journals.The tetracyclines are the most complex of the

chlorinated structures. Six analogues of chloro-tetracycline are known to be of biosynthetic ori-gin. Oxytetracycline is not included here since, ina biological sense, it is not derived from chlor-tetracycline, but is included in the table becauseof chemical and certain biological relationships.Other tetracyclines, such as the epi-forms, are notincluded because they are not of biosyntheticorigin.

Sclerotioramine has been reported to be anatural product (34) since it was isolated fromcultures of Penicillium multicolor which had beenincubated for a longer than normal time period.However, since sclerotiorin is known to be verysusceptible to amination, and since P. multicoloris a notable producer of ammonia in older culture,sclerotioramine was more likely a result of theaction of ammonia on sclerotiorin rather than adirect product of the organism. For this reasonsclerotioramine is not included in table 1.

It is rather interesting that halogen-containingcyclic organic compounds are of more commonoccurrence, or knowledge, than aliphatic types.This may reflect a result of ease of detection sincecyclic compounds are common organic pigmentsor have biological activity, thus facilitating iso-lation.

B. BiologicalWith the exception of some limited information

concerning certain chlorinated anthraquinonepigments of flowers, apparently there is a generaldearth of knowledge concerning halometabolitesin the higher plants. Metabolism of chloride asmeasured by its uptake or effects on growth arenoted, but what happens to the chloride is notdisclosed. It seems unusual to find information onhalometabolites limited to the Thallophyta. Thelimited number of genera and species which pro-duce halometabolites is indicated in table 1. Thetaxonomic positions are scattered. In the Schizo-mycetes, the Eubacteriales are represented bythe genus Pseudonwnas, and the Actinomycetalesby the genus Streptomyces. Three orders are repre-sented in the Ascomycetes; the Lecanorales byspecies of Buellia, Lecanora, Pannaria, Parmelia,Pertusaria, and Evernia; the Pezizales by thegenus Mollisia; and the Aspergillales by the

species of Aspergillus and Penriillium. The genusPsathyrella represents the Basidiomycetes. Lastly,the genus Caldariomyces represents the FungiImperfecti. Other genera reported to metabolizechloride include Absidia, Alternaria, Botrytis,Cephalosporium, Cladosporium, Clasterosporium,Helminthosporium, Mucor, Stemphylium, Sty-sanus, Syncephalastrum, and Trichoderma (21).This metabolism was indicated by the uptake ofchloride from the substrate, but no specific halo-metabolites were characterized.

Generally neglected in previous literature cita-tions on fungus metabolites, the lichen metabo-lites are here considered to be true fungal prod-ucts. This view is substantiated by the fact thatthe depside nuclei are known to be products oflichens (21) and of fungi (79); the depsidonenuclei are produced by lichens (80) and by cul-tured fungi (88). Halogenated depsidones areknown to be products of both cultured fungi (27)and of lichens (5). Further, laboratory culture ofthe fungal component of a lichen, Cladoniacristatella, produced the same characteristic or-ganic acids produced by the lichen in nature (17).All indications are that these characteristic or-ganic compounds are the products of the fungalcomponent of the lichen. Therefore, it seemsreasonable to give Zopf (120) credit for findingthe first fungal halometabolite, diploicin (IX), inthe lichen Buellia canescens.

It is not uncommon to find reports of a commonorganic nucleus biosynthesized by a number ofspecies in the achlorophyllous thallophytes.These common nuclei, however, are usually foundonly within certain taxonomic limitations. Inconsidering the halometabolites (table 1 andfigure 1), common molecular structures are pro-duced by different genera in the case of the dep-sidones; spirans are produced by species of Asper-gillus and Penicillium; the genus Aspergillus pro-duces both depsidone and spiran derivatives.Specifically, groups 8, 9, and 10 of table 1 appearto be very common products of the ascomycetousfungi. In these cases, the microorganism hadbeen described prior to the finding of the specifichalometabolite concerned. Here reduction of theearlier used specific names to synonymy has oftenoccurred. For example, Penicillium janczewskiihas been reduced to synonymy with P. nigricans,and P. griseofulvum to P. urticae. Misidentifica-tion is not uncommon as, for example, the culturealleged to be Aspergillus ustus, the producer of

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ustin, i.e., nornidulin (table 1: 9e), was later re-identified as Aspergillus nidulans (27).A number of binomials are associated with

specific halometabolites of Streptomyces origin.This may reflect among other things a failure tosurvey critically the known literature and to com-pare critically the new culture with related types(86). One cannot help having the impression thatthis is often applicable in patent documents,especially when the description of the alleged newspecies is excessively supplied with trivial infor-mation but inadequate with respect to presentlyaccepted major taxonomic characteristics. Thecritical investigator is often at a loss with theseproposed names, first through lack of essential in-formation, and second, through lack of parallel

laboratory comparisons when type-culture ma-terial is not publicly available.

Chloramphenicol (Table 1: 4a) is produced byS. venezuelae (37). Streptomyces omiyaensis (110)was described as producing chloramphenicol, butno reference was given as to whether the type wascompared with S. venezuelae, the distinction beingmade on the basis of lack of brown pigment inprotein media and lack of curved sporophores;however, the sporophores were not described.They were later noted to be flexuous (86). At thattime Umezawa et al. admitted the similarity ofStreptomyces phaeochromogenus var. chtoromy-ceticus to S. venezuelae, and the former was re-duced to synonymy by Waksman | rd Leche-valier (114). The multiplicity of legitimate

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MILTON A. PETTY

species producing chloramphenicol has yet to beestablished.

Chlortetracycline (Table 1: 12e) is producedby S. aureofaciens (32), but certain cultures ofthis species are not obligate halogenators of thetetracycline (Table 1: 12h) nucleus (30, 59, 115)since isolants from nature as well as mutantsfrom chloride-utilizing strains may producetetracycline in addition to chlortetracycline evenin the presence of excess chloride. It has not beenour experience to find natural isolants of Strep-tomyces capable of producing tetracycline but in-capable of producing chlortetracycline. Everynatural isolant encountered here which wascapable of producing tetracycline in any appre-ciable proportion could be classified as S. aureo-faciens even when the property of antibioticproduction was disregarded. Therefore, speciesalleged to produce tetracycline should be examinedcritically for identity with S. aureofaciens. Strep-tomyces viridifaciens ATCC 11,989 (45) is knownfrom personal experience to make chlortetracy-cline in a conventional fermentation medium con-taining chloride, e.g., a fermentation system suchas described by Goodman et al. (41) containing1000 ppm chloride. This culture yielded 1.06g/L total tetracyclines with a chlortetracyclineto tetracycline ratio of 8.9:1. In addition, S.viridifaciens conforms to accepted specific cri-teria of S. aureofaciens (E. J. Backus, personalcommunication; (6); author's experience) and insusceptibility to the monovalent S. aureofaciensactinophage (R. Weindling, personal communica-tion). Therefore, specific rank is believed not jus-tified for S. viridifaciens.

S. aureofaciens ATCC 11,926 is described asproducing quinocycline, an antiboitic distinctfrom tetracycline and not containing halogen (18).Streptomyces fuscofaciens ATCC 12,061 (19) isalleged to produce quinocycline and tetracyclinein media low in chloride. This British Patentstates ". . . if the chloride ion content of themedium is reduced to a minimum, the proportionof tetracycline to other broad spectrum anti-biotics is favorably affected." This indicates thatS. fuscofaciens has characteristics in commonwith S. aureofaciens. Further, only one interpre-tation is known to be possible concerning reduc-ing the proportion of "other broad spectrumantibiotics" in a tetracycline fermentationthrough the denial of chloride to that fermenta-tion, namely, in the presence of chloride, chlor-tetracycline would be formed in greater propor-

tions. The obvious conclusion is that cultureATCC 12,061 produces chlortetracycline! Lackof description of certain critical characters andlack of public availability of culture ATCC12,061 make it impossible to confirm either thejustification for the new epithet, or whether itdoes or does not produce a halometabolite.

Generally overlooked in the literature, Strep-tomyces sayamaensis (94, 95) is disclosed as pro-ducing ehlortetracycline and tetracycline. Thesepublications do not present a description of theculture, but this is only later found in a patent(4). A thorough study of this culture has showna majority of specific and critical criteria in com-mon with S. aureofaciens (E. J. Backus, personalcommunication; author's unpublished data), in-cluding sensitivity to the specifically monovalentS. aureofaciens actinophage (R. Weindling, per-sonal communication). Therefore, it appears neces-sary to reduce S. sayamaensis to synonymy withS. aureofaciens.

Considering then whether chlortetracyclineand tetracycline are the products of multiplespecies-and the evidence does not bear this out-and despite the fact that these four binomialshave been proposed, although only two have beenused by authorities in the scientific literature,only one of these two species appears to have metthe criteria of valid publication, and that is S.aureofaciens. Other allegedly different taxonswhich have been proposed solely for the produc-tion of tetracycline will be noted below.An interesting sidelight is the fact that although

Streptomyces rimosus and Streptomyces hygro-scopicus can produce oxytetracycline, neither ofthese is indicated as capable of producing ahalogenated tetracycline.

III. FACTORS INFLUENCING BIOSYNTHESIS OF

HALOMETABOLITES

A. Genetic

Strain improvement is in part a term borrowedfrom agricultural genetics. It denotes the obtain-ing of superior culturable stock. Of the knownhalometabolite producers, only S. aureofacienshas received much attention in the literature inthis respect. The original strain of Duggar,A-377, produced 20 to 100 4g/ml of chlortetra-cycline. The first report of strain improvementwith this microorganism recorded a high yield of1300 ,ug/ml (113). In a series of mutation treat-ments, a strain has been derived by Growich (30)

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which is said to produce a yield of 8.5 g/L oftotal tetracyclines (42). A comprehensive treat-ment of genetic manipulation of S. aureofaciensis presented by Alikhanian et al. (1). After a pre-liminary treatment of spores with ethyleneimine,ultraviolet irradiation gave a threefold increaseover the frequency of morphological mutationsachieved with ultraviolet light alone. An increasedmutation rate coupled with an increased survivalof spores resulted when irradiation with X-rayswas followed 2 hr later by ultraviolet irradiation.The reverse sequence of irradiation or the sole useof one type of irradiation was not nearly aseffective. In the selection of strains yielding highchlortetracycline to tetracycline ratios, a five-stage selection procedure gave strains of con-siderably increasing antibiotic-producing ability.

Strain-improvement procedures often have in-teresting and important side effects, such as re-warding the investigator with new analogues ofthe parent compound. Such was the case with thepurely laboratory-derived demethyltetracyclines(72), and chlorodehydrotetracycline (71). Spon-taneous variation in a strain of Aspergillus terreusproducing geodin-erdin (XIV) resulted in theproduction of geodoxin (XIII) according to Has-sal and McMorris (49).

Strain-improvement techniques have affordedproduction of deshalo-analogues even in thepresence of available chloride ion. This is termedgenetic control of halogenation, which here indi-cates the induction of variation in the ability of aculture to halogenate an organic metabolite. Ithas been known for some time that differentstrains of A. terreus differed in their ability tometabolize chloride as measured by the disap-pearance of chloride from the substrate (21), butthere was no indication that any of them were orwere not producing the same halometabolite or adeshalo-analogue. In fact, a deshalo-analogue ofa halometabolite of A. terreus has yet to be re-ported. The first indication that different strainsof the same species differed in their ability toproduce the deshalo-analogue of a halometabolitewas with P. nigricans, of which one culture pro-duced no dechlorogriseofulvin, another cultureproduced dechlorogriseofulvin in a ratio range ofabout 1:20 to griseofulvin, and a third had a ratiorange of about 1:4 (65). In the formation ofchlortetracycline, S. aureofaciens is known to bea remarkable scavenger of chloride (31) even tothe extent of its being suggested as a tool for theassay of chloride-containing materials (55).

Sekizawa (93) reported an attempt to controlhalogenation of tetracycline genetically, but wasunsuccessful. Shortly thereafter, Martin et al.(68) indicated that this could be done. Theystated, "Both natural and induced mutants of S.aureofaciens may produce tetracycline in varyingamounts in addition to other antibiotics. Byselection of a natural or induced mutant whichproduces a comparatively high ratio of tetra-cycline to other antibiotics present, . . . andpropagating the organism..., a fermentativemash results from which tetracycline may beeconomically recovered." In this patent areexamples showing the tetracycline production inthe presence of available chloride. These allega-tions were substantiated by Doerschuk et al. (30),who showed that the S. aureofaciens of Duggarand most of its progeny would halogenate thetetracycline molecule at a constant rate, inde-pendent of chloride concentration. These con-stant utilizers could be termed chloride scaven-gers, for at low chloride concentrations they couldbe used as analytical tools for the detection oftraces of chloride. In addition, Doerschuk et al.described a second class of strains, originatingfrom the first and halogenating the tetracyclinemolecule at a reduced and inconstant rate, whichwas dependent upon chloride concentration. Thefirst and second classes differed in their behaviorwith respect to bromine and halogenation in-hibitors. By experience, natural isolants of S.aureofaciens preferentially halogenate the tetra-cycline molecule, and only rare isolants produceany appreciable proportion of tetracycline in thepresence of available chloride. With normalchloride-utilizing strains, tetracycline is syn-thesized at the expense of chlortetracycline.More recently other workers have reported

strains of S. aureofaciens which were not chloridescavengers. A natural isolant and induced mu-tants of S. aureofacies were found to producetetracycline in the presence of available chlorideby Wang (115). From a normal chloride-scaveng-ing strain, an inconstant utilizer was derivedwhich produced a chlortetracycline to tetracy-cline ratio of 1:4 in a fermentation yielding a totalpotency of 1800 to 2000 ,g/ml and containing anexcess of available chloride ion (59). Mutants ofS. aureofaciens "with considerably reducedchlorinating activity, insignificant in comparisonwith that of the parent strain," have been ob-tained by Kollar and Jarai (56). The strains usedby both Kotiuszko et al., and Kollar and Jarai

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were of the same parental stock; t.e., SovietLS-536. That no absolute chloride-ignoring strainhas been isolated from nature confirms our ex-perience. However, a laboratory-derived mutantstrain 4623/33, alleged to be that described in aBritish Patent Specification (62) appears to ap-proach closely the point of completely lacking afunctional chlorinating mechanism.The ability of S. aureofaciens to halogenate a

metabolite may thus be impaired by conventionallaboratory mutagenic treatments, and geneticcontrol of halogenation is thereby affirmed.

B. Chemical

In the formation of a halometabolite, availablehalide is obviously necessary. On the other hand,halide denial to a halometabolite producer doesnot assure the formation of the correspondingstrict chemical deshalo-analogue (24, 52, 101).Dean et al. (24) reported that a halometabolite,griseofulvin, was obtained when Penicilliumbrefeldianum was grown on Czapek-Dox, achloride-containing medium; but instead ofgriseofulvin an unrelated and nonhalogenatedsubstance, fulvic acid, was obtained when thisspecies was grown on a Raulin-Thom mediumwhich does not contain deliberately added chlo-ride. Jackman et al. (52) did not find deschloro-selerotiorin (VI, R==H) produced by Penicilliumsclerotiorutm in a Raulin-Thom medium, butrather a substance of altered chemical configura-tion which they termed rotiorin (VII). Rotiorinwas produced in larger quantity along withsclerotiorin on Raulin-Thom medium than onCzapek-Dox medium. Smith (101) found anunexpected alteration of synthesis by a chloram-phenicol-producing Streptomyces strain whenhalide was withheld. Instead of obtaining only thedeshalo-analogue of chloramphenicol, in additionhe obtained the N-propionyl- and N-butyryl-derivatives of p-nitrophenylserinol in amountsconsiderably in excess of what had been expected,based on the yield of chloramphenicol. Neithercan it be said that the lack of halides deliberatelyadded to conventional substrates precludes theformation of halometabolites. For example, Brianet al. (15) produced the curling factor, griseoful-vin, on Raulin-Thom and corn steep media,neither of which had deliberately added halides;nalgiolaxin was encountered in cultures grown onRaulin-Thom solution (89); and chlortetracyclinewas first produced in a fermentation not furtherenriched with added halide (33). By definition,

all microorganisms capable of synthesis of halo-metabolites must be able to metabolize ionichalide. Halopenicillins, which are achieved byfeeding a halogenated precursor to the fermenta-tion, are not halometabolites. These will not beconsidered here.Only two substances, copper and fluoride,

appear to be functional in promoting halogena-tion. In a patent (105) the ratio of chlortetra-cycline to tetracycline is said to be doubled in thepresence of fluoride under certain conditions.Copper, which has long been recognized as func-tional in oxidative enzyme systems, is apparentlyfunctional in microbial halogenation (41, 93).The behavior of copper will be dealt with morefully under the section on Inhibition of Halogena-tion. Generalizations on the essentiality of copperfor microbial halogenating enzymes becometenuous in view of the results of Galliechio andGottlieb (40) which indicate that biosynthesis ofchloramphenicol-like activity was increased whencopper was eliminated from a synthetic medium,and instead, that biosynthesis of such activitywas stimulated by the presence of cations ofmagnesium, iron, and zinc.There are no specific organic chemicals reported

which favor halogenation. Specific organic com-pounds favoring synthesis of specific metaboliteshave been reported. For example, quinic acid andshikimic acid are reported as precursors in thebiosynthesis of the tetracyclines (45). However,this precursor effect was not confirmed by inde-pendent investigators (74) who found only aninsignificant amount of radioactive chlortetracy-dline after addition of shikimic-C'4 acid to a fer-mentation which was rapidly synthesizing chlor-tetracycline. A number of organic compounds arereported by Vaneik (112) to stimulate the syn-thesis of chlortetracycline by a low yielding strainof S. aureofaciens. Among others, phenylaceticacid, fl-indolylacetic acid, p-chlorophenoxyaceticacid, a-naphthylamine, a-naphthylacetic acid,and p-aminobenzoic acid were active. In our ex-perience with better yielding strains, these orsimilar type compounds have not effected stimu-lation as reported by Vanek. Certain chemicalshave been reported as stimulating the biosyn-thesis of chlortetracycline. Benzyl thiocyanateat 0.5 mg/L stimulated the formation of as muchas 800 mg/L chlortetracycline above a 1700 mg/Lcontrol yield (84). The mechanism of this actionis not known, but it may be assumed to be in al-tering carbon metabolism (50). Egorov and

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Baranova (35) reported stimulation by paradi-methylaminobenzaldehyde, but from the datapresented it is not possible to deduce whetherthis compound is acting as a stimulant or as aprecursor for ring A (figure 1, XVII).

Salts of bivalent cations such as calcium, mag-nesium, and strontium, have a very desirableeffect in the chlortetracycline fermentation sys-tem in that they precipitate or complex the anti-biotic into an insoluble form and away from thepresence of the formative microorganism (77).In fermentation mashes containing an alkali re-serve of CaCO3, less than 100 ,ug/ml of antibioticis present in the mash filtrate, which is a levelwell tolerated by the S. aureofaciens strain used.When the concentration of antibiotic goes above100 gAg/ml in the filtrate, evidence of inhibition ofthe formative microorganism begins to appear.The highest publicly disclosed total tetracyclinesyield is 8.5 g/L (42). On a material-balance basis,this indicates that at least 7 per cent of the totalcarbon of the nutrient medium has been con-verted into tetracyclines. If 80 to 90 per cent ofthe tetracycline carbon arises from the starch(74), then the carbon conversion is approximately8 to 9 per cent; in these fermentations, themycelial dry weights are in the range of 30 to 40g/L, and this suggests that the mycelium iscapable of the synthesis of a fifth to a fourth ofits own weight of tetracyclines.The concentration of phosphorus compounds in

the fermentations by S. aureofaciens influencesthe amount of chlortetracycline formed. Biffi et al.(8), using a corn steep formula, showed that theaddition of phosphate radically changed themetabolic behavior of S. aureofaciens in thatribonucleic acid content of the mycelium, sucroseand ammonia consumption, and pyruvic acidaccumulation were increased, whereas chlor-tetracycline synthesis was sharply diminished.It seems that the carbon of the sucrose wasbeing accumulated as pyruvic acid rather thanbeing used in the synthesis of chlortetracycline.Other workers have confirmed these observa-tions (87).

C. Inhibition of HalogenationOne of the most interesting developments

concerning halometabolites is the field of chemicalinhibition of halogenation. MacMillan (66) un-intentionally presented the first data to thiseffect when he reported that strain no. 250 ofP. nigricans, which normally produced no de-

chlorogriseofulvin, produced a mixture of bromo-analogue and dechlorogriseofulvin when grownin a bromide-rich, chloride-poor medium. Thefirst purposeful disclosure was by Sekizawa (93),who found that bromine and four thio- ormercapto- compounds were effective in blockingthe incorporation of chloride ions in a chlor-tetracycline fermentation. The inhibitory effectof bromide was confirmed (3, 46). Bromide ionsact as a competitive inhibitor; i.e., if one moleculeof Br- will block one molecule of Cl-, or if theratio of Br- to Cl- were 0.5, then the ratio oftetracycline to total tetracyclines would be 0.5.Thiocyanate, which is also classed as a competi-tive inhibitor, was not incorporated into thetetracycline molecule in contrast to bromide,which was incorporated to form bromotetra-cycline (Table 1: 12b) (31). The action of com-petitive inhibitors may be reversed by excess ofthe normally utilized halide (31). These actionshave an analogous parallel in the animal thyroidgland's uptake of iodine and its inhibition. Ofeven more interest were the enzyme inhibitorswhich were dealt with more fully by Lein et al.(61), and Goodman et al. (41). The best in-hibitors in the system of Lein et al. were 2-benzoxazolethiol; 2, 5-dimercapto-1,3, 4-thiadi-azole; and 2-mercaptobenzimidazole. Goodmanet al. have confirmed the inhibitory effect of thesecond compound of Lein et al. and add 2-phenyl-5-mercapto-1,3, 4-oxadiazole and 2-(2-furyl)-5-mercapto-oxadiazole to the list. One of the moreremarkable aspects of these enzyme inhibitors isthe minute amount required to block the chlori-nating system. As little as 5 Ag/ml of inhibitor wasable to block the incorporation of 400 4g/ml ofchloride into the tetracycline molecule. The dataof Goodman et al. may be considered a rigoroustest of inhibition since they were working with ahigh potency system which was enriched withchloride and with a known "chloride scavenging"strain. The action of these inhibitors appeared tobe, at least in part, associated with a function ofcopper, probably a copper oxidase, since theaddition of copper reversed the effect of theinhibition. The addition of excess chloride didnot reverse the inhibition. Interestingly, silverion was able partially to reverse the inhibitioneffect.At present, data on the chemical inhibition of

halogenation have not been extended to otherhalometabolites except chloramphenicol andcaldariomycin. In chloramphenicol fermentation,

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bromide was competitive with chloride (101),whereas fluoride or iodide had no effect. Anumber of chlorination inhibitors is indicatedby Shaw and Hager (98) for Caldariomycesfumago in the synthesis of caldariomycin; sodiumazide was the most potent, followed by thiourea,thiouracil, and 6-methylthiouracil. Inhibition toa lesser degree was indicated for bromide,iodide, and fluoride. Because of conflicting dataon inhibitors, Shaw and Hager were unable toconclude that a copper or heme oxidase wasimplicated in this chlorination reaction. Theirchlorination system was relatively insensitiveto cyanide but highly sensitive to azide in-hibitors. Copper oxidases should be affected byboth of these two types of inhibitors. A stimula-tion of formation of tetracycline in a chlor-tetracycline fermentation has been attributed toincreasing the concentration of corn steep liquorin the medium (78). However, from the datapresented, it is difficult to interpret this effectas an inhibition of chlorination, or as an increasein the total potential of a system above itschlorinating capacity.

IV. HALOGEN INTERCHANGEABILITY

No workers have reported iodo- or fluoro-metabolites of microbial origin although attemptshave been made to produce iodo- and/or fluoro-metabolites of A. terreus (88), of P. sclerotiorum(91), of S. aureofaciens (30), and of a chlor-amphenicol-producing Streptomyces (101). Onlyfive bromo-derivatives are known: bromo-de-chlorogriseofulvin (Table 1: 10f) (66); bromo-tetracycline (Table 1: 12b) (96); N-dibromo-acetyl-p-nitrophenylserinol (Table 1: 4d) andN-monobromomonochloroacetyl-p-nitrophenyl-serinol (Table 1: 4e), the analogues of chloram-phenicol (101); and bromo-demethyltetracycline(Table 1: 12a) (70).Although bromide interchanges with chloride,

it is incorporated by the microorganisms in-volved at levels much lower than those recordedfor chloride. For instance, P. nigricanrs strainno. 250, which did not produce dechlorogriseo-fulvin in chloride-rich media, when grown in achloride-limited, bromide-rich medium, produceda mixture which, after extraction, yielded aratio of 62:73 of bromo- and dechloro-analogues(66). S. aureofaciens of the type which normallyproduced chlortetracycline in chloride-rich media,when grown in a chloride-limited, bromide-richmedium produced a mixture which, after extrac-

tion, was composed of approximately nine partsof tetracycline to six parts of bromotetracyclineas calculated from countercurrent distributiondata of Sensi et al. (96). The high yielding mutantof S. aureofaciens (31), BC41, brominated atonly one-third of its chlorinating rate. Thiscompares favorably with the calculations madefrom data of Sensi et al. Biologically, the bromo-analogues seem to offer no gross advantages overthe chlorometabolites as antibiotics. Bromo-dechlorgriseofulvin is only one-seventh as activeas griseofulvin (66). Bromotetracycline hasessentially the same spectrum and level ofactivity as does chlortetracycline (96) and thebromo-analogues of chloramphenicol do nothave a high order of activity against Klebsiellapneumoniae (101).

V. BIOSYNTHESIS AND MECHANISMOF HALOGENATION

In the biosynthesis of penicillin, relativelylarge precursor fragments of the ultimate moleculeare incorporated by Penicillium. This fact hasmisled many investigators in the search foranalogous and economically attractive parallelsin other valuable microbial metabolites. Birchet al. (9) have given evidence for the buildupof sclerotiorin from acetic acid units, and thistheory of biosynthesis has support in studies onother microbial metabolites.

In separate experiments, by growing P.multicolor in media containing radioactive car-bon-labeled acetic acid, both for the methyl andcarbonyl carbon, and formic acid, Birch et al.(9) obtained different radioactive sclerotiorins.After degradation of these forms and measuringthe radioactivity of different fragments, theyconclude that the skeleton of sclerotiorin isformed from linkage of 9 molecules of aceticacid and 3 molecules of formic acid. Theyrepresent the origin of the molecule as:

C1

0.-A .... A.....-A* ~ ~~~~+*1

,

V--+A A 0 V V

(Symbols: * carboxyl carbon of acetic acid;A = methyl carbon of acetic acid; V = carbonof formic acid. Compare with formula VI offigure 1.) They also indicated through radio-

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carbon studies that a possible precursor, a

dienoic acid, was degraded to acetic acid by thegrowing culture prior to being incorporated intothe sclerotiorin molecule.

Earlier Birch et al. (11) presented evidence,using carbonyl C'4-labeled acetic acid, for thebiosynthesis of griseofulvin by a similar acetatelinkage. They represent the origin of griseofulvin,arising from 7 molecules of carboxyl carbon-labeled *acetic acid, as:

0 0

C-C

CI D *COC

(Compare with formula XV of figure 1.) Sincethe molecules of geodin and erdin (XLV) havethe same skeleton as does griseofulvin, it may

be assumed that their mechanism of origin issimilar to that of griseofulvin. The same mightbe said for geodoxin (XIII), except that oxygen

must be introduced between the carbons ofone of the acetate groups in the linkage sequence

postulated by Birch et al. for griseofulvin. It wouldbe very interesting to see the same type ofstudy on the products of A. terreus as has beendone with those of P. multicolor. Since both ofthese species will produce these metabolites on

media containing only glucose as an organiccarbon source, they must be able to produceacetic acid and methyl donor compounds bytheir own metabolism.The simplest halometabolite, caldariomycin,

has received recent attention from Shaw, Beck-with, and Hager (97-100). They first demon-strated that ,B-ketoadipic acid and chloride were

converted into chlorolevulinic acid by driedmycelial powders of C. fumago. The stoichiometryof this reaction was represented as follows:

O-Ketoadipic acid + C1- + A2 02 + 2H+)- CO2+ H20 + chlorolevulinic acid.

The pH optimum of the reaction was 4.8,consistent with the involvement of hydrogen ion.There was a high rate of oxygen uptake andcoincident carbon dioxide evolution. Chlorina-tion was not accomplished in the absence ofoxygen. ,B-Ketoadipate was slowly oxidized inthe absence of chloride, but without carbondioxide production. The hydrolysis of a polysac-

charide, present in considerable amount andresistant to attack by amylolytic enzymes, wasdeduced to be responsible for the high endogenousoxygen uptake by the reaction.

In later studies Shaw et al. separated twofractions from an extract of mycelial powders,(a) a heat-stable supernatant and (b) a heat-labile gel eluate, both of which were required forformation of chlorolevulinic acid. The heat-stable fraction contained a polysaccharide, andmild acid hydrolysis of this fraction enhancedits activity. Glucose and hydrogen peroxidewould substitute for the heat-stable fraction.Further separation of the heat-labile fractionyielded two components, which were called: (c)a glucose oxidase fraction, and (d) a chloro-peroxidase fraction. Both of these fractions werenecessary for the synthesis of chiorolevulinicacid from chloride and /3-ketoadipate in thepresence of glucose. They formulated the reac-tions involved.

(Glucose). + 3H20

-hydrolase (glucose).-3 + 3 glucose3 Glucose + 302 + 3H20

glucose3 gluconic acid + 3 H202 + 3H+

oxidase

2 H02 catalase 02+ 2 H20H,02 + Cl-

+ -OOC-CH2-CO-CH2-CH2-COO-

+ 2 H+ chloroperoxidase C02+ C1CH2-CO-CH2-CH2-COO-+ 2HO20

In the last reaction, they suggested that chlorideion was oxidized to a chlorinium ion with oxygenserving as an electron acceptor. They found noevidence to support the participation of anenergy source such as adenosine triphosphate,except that there was a high rate of endogenousoxygen uptake. The fact that decarboxylation offB-ketoadipate did not proceed in the absence ofchloride was evidence to them that an enzymat-ically generated free enol form of levulinic acidcould not be the chlorine acceptor.The isolated components of this system did

not form caldariomycin. There was an 8 percent conversion of the radioactive chlorine of-y-chlorolevulinic acid to caldariomycin by activecultures of C. fumago without evidence that

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incorporation proceeded via hydrolysis of thesubstrate to yield chloride ion. It appears thatthe introduction of the second chlorine of calda-riomycin is accomplished by a separate series ofreactions and that the biogenesis of caldariomycinmay be hypothesized to involve the cyclizationof -y-chlorolevulinic acid.

Cl

OH CH2

O=C C-=O

bH2 -H

Cl H

C

O=C C=-- I

CH2- H2

Knowledge of the biosynthesis of chlor-amphenicol is limited (43). S. venezuelae ap-parently does not chlorinate N-acetyl-p-nitro-phenylserinol (44), for when p-nitrophenylserinolwas added to cultures it was acetylated, butnot acetylated and chlorinated.

O H2COH

N -C-C-NH2 + CH3COOH431 H

O 0H

C

C

H2COHH I

N- -C-C-N-C-CH3 + H201/ H H 11

0

H

The results of Wang et al. (118) led them tosuggest that a possible precursor, dichloroaceticacid, is first converted into an intermediateprior to being incorporated into the chlor-amphenicol molecule. Their results did notindicate the prior conversion of organic chlorideto ionic chloride except in the case of mono-

chloroacetic acid.In the biosynthesis of tetracyclines by S.

aureofaciens, chlorination apparently did notoccur in the later stages (46), but neither didit occur at the very beginning (31), because theover-all rate-limiting step preceded the halogena-tion step. Evidence that halogenation was notthe final step is the fact that addition of tetra-cycline to a chlorinating system did not resultin chlorination of the added tetracycline (115).

Wang (116, 117) has isolated but not charac-terized organic chlorine-containing compoundsfrom the young liquid cultures of S. aureofaciens.Three of these chlorine-containing organic frac-tions were tested for incorporation into chlor-tetracycline by radiochemical methods. Onlyone, called the "aqueous soluble chlorine com-pound," which was isolated from culture filtrate,was indicated to be incorporated. The other twowere incorporated into the mycelium but notinto chlortetracycline. It would appear fromboth the results of Wang and Doerschuk et al.(31) that the metabolized chloride is eitherincorporated into antibiotic or into myceliumbecause chlorine-containing organic compoundsin the filtrate at the end of the fermentationwere not observed.The observation that the 7-chloro-5a(lla)-

dehydrotetracycline can be biologically or chemi-cally hydrogenated, thus yielding chlortetra-cycline (71, 73), led to the suggestion that thishydrogenation is the last step in the biosyntheticpathway.C1 CH3 OH H N(CH3)2

-OH + H--C-NH2

HC1 CH3 OH H N(CH3)2

/H|HOH

-C-NH2

0 0 O H 0H H

The dehydro compound, which had a low orderof antibacterial activity in vitro, was producedby a mutant of S. aureofaciens. The presentlyfavored proposal for the biogenesis of the basicnaphthacene nucleus of chlortetracycline is bythe "head to tail" linkages of molecules of aceticacid as first theorized for oxytetracycline byRobinson (92).

Apparently a remarkable series of enzymaticreactions is involved in the biosynthesis of thechlortetracycline molecule from simple begin-nings. Radio-carbon was incorporated into thetetracycline skeleton by S. aureofaciens from2-, 3-, 6- and poly-carbon nutrients (74). Theseauthors show the specific incorporation of the2-carbon of glycine, the methyl carbon of

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methionine, the 1- and 2-carbons of acetate,and the 3-carbon of serine. The high percentageof incorporation of the 2-carbon of glycine andthe methyl group of methionine indicated a

rather direct route of incorporation. The almostequal incorporation of the 1- and 2-carbons ofacetate indicated the use of the whole acetategroup. The marked difference of incorporationof the carbons of glycine indicated that this was

serving as a donor of 1-carbon groups. Degrada-tion of chlortetracycline synthesized by a fer-mentation containing glycine 2-C"4 indicated a

concentration of radioactivity in the dimethyl-amino group of the molecule. Nonradioactiveglycerol was said to furnish all the carbonrequired for the germination of spores, productionof mycelium and formation of chlortetracyclineby S. aureofaciens in a mineral salts medium.

If we postulate the origin of chlortetracyclinefrom acetate linkages, it is more probable thatthe 7-carbon of chlortetracycline-that is, thecarbon of the chlorine-carbon bond-originatesfrom the methyl carbon of an acetate group.

This has its parallel in other halometabolites(9-11). It is also reasonable to expect that the1-, 10-, 11-, and 12-carbons of the moleculeoriginate from carbonyl carbons of acetategroups. After linkage and cyclization of theacetate groups, a polyketone skeleton is organized.

C C C C

CO \CO \CO CO CO

C0 C0 C0 C0 C

\ \ \ /\ /CO CO CO CO

At least 12 other reactions must be accomplishedto complete the molecule: (a) addition of a

-CONH2 group at the 2-carbon; (b) hydroxyla-tion of the 3-carbon; (c) amination of the 4-carbon with synchronous or subsequent methyla-tion of the amine; (d) and (e), respectivereduction of the 4a-, 5a-carbons and their hydro-genation; (f) and (g), respective hydroxylationand methylation of the 6-carbon; (h) chlorina-tion of the 7-carbon; (i) reduction of the 8-car-bon; (j) and (k), respective enolization of the10- and 12-carbons; and (1), the hydroxylation ofthe 12a-carbon, which may be coupled with thereduction of the 4a-carbon. The sequence ofthese events is not known, except as mentionedearlier, reaction (d) may be one of the last tooccur.

Thus a common basic mechanism of synthesis

is indicated for a growing list of metabolites,but with infinite variations and terminationsaccording to the species and its environment.There is insufficient evidence for generalizationsconcerning common mechanisms of halogenation,but instead the available evidence indicatesdifferences in mechanisms.

VI. FUNCTION OF HALOMETABOLITES

None of the halometabolites described hasbeen assigned an essential function in themetabolism of the microorganism that produces it.Functionally, the antibiotic metabolites mayserve to give the specific producer moreLebensraum than it would be afforded in ordinarycommensal relations, but this effect has yet tobe demonstrated in nature. The close similarityof certain halometabolites to certain antimetab-olites; i.e., vitamin K, its dichloro-antimetab-olite, and diploicin- causes certain teleologicalspeculations that these products are protec-tive mechanisms. However, until more knowledgeis amassed on the metabolism of chloride and ofhalometabolites, the attitude must be held thathalometabolites are unusual and chance productsof nature.

VII. REFERENCES1. ALIKHANIAN, S. I., MINDLIN, S. Z., GOLDAT,

S. I., AND VLADIMIZOV, A. V. 1959 Gen-etics of organisms producing tetracyclines.Ann. N. Y. Acad. Sci., 81, 914-949.

2. ANCHEL, M. 1952 ldentification of droso-philin as p-methoxytetrachlorophenol. J.Am. Chem. Soc., 74, 2943.

3. ARISHIMA, M., SEKIZAWA, Y., SAKAMOTO, J.,MIWA, K., AND OKADA, E. 1956 On thetetracycline fermentation. J. Agr. Chem.Soc. Japan, 30, 407-409.

4. ARISHIMA, M. AND SEKIZAWA, Y. 1959 Amethod of preparing tetracycline. Japan-ese Patent No. 255,818. Japanese PatentPublication No. 4798/59.

5. ASAHINA, Y. AND SHIBATA, S. 1954 TheChemistry of lichen substances. Japan Soc.for the Promotion of Science, Tokyo.

6. BACKUs, E. J., DUGGAR, B. M., ANDCAMPBELL, T. H. 1954 Variation inStreptomyces aureofaciens. Ann. N. Y.Acad. Sci., 60, 86-101.

7. BARTON, D. H. R. AND SCOTT, A. I. 1958The constitutions of geodin and erdin. J.Chem. Soc., 1958, 1767-1772.

8. BIFFI, G., BORETTI, G., DIMARCO, A., ANDPENNELLA, P. 1954 Metabolic behavior

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and chlortetracycline production by Strep-tomyces aureofaciens in liquid culture.Appl. Microbiol., 2, 288-293.

9. BIRCH, A. J., FITTON, P., PRIDE, E., RYAN,A. J., SMITH, H., AND WHALLEY, W. B.1958 Studies in relation to biosynthesis.XVII. Sclerotiorin, citrinin, and citromy-cetin. J. Chem. Soc., 1958, 4576-4581.

10. BIRCH, A. J. AND MASSY-WESTROPP, R. A.1957 Studies in relation to biosynthesis.XI. The structure of nalgiovensin. J.Chem. Soc., 1957, 2215-2217.

11. BIRCH, A. J., MASSY-WESTROPP, R. A.,RICKARDS, R. W., AND SMITH, H. 1958Studies in relation to biosynthesis. XIII.Griseofulvin. J. Chem. Soc., 1958, 360-368.

12. BIRKINSHAW, J. H. 1952 Studies in thebiochemistry of microorganisms. 89. Meta-bolic products of Penicillium multicolorG.-M. and P. with special reference tosclerotiorin. Biochem. J., 52, 283-288.

13. BRACKEN, A. 1954 Naturally occurringchlorine containing organic substances.Mfg. Chemist, 25, 533-538.

14. BREEN, J., KEANE, J., AND NOLAN, T. J.1937 The chemical constituents of lichensfound in Ireland. Pertusaria concreta Nyl.form Westringii Nyl. Sci. Proc. Roy.Dublin Soc., 21, 587-592.

15. BRIAN, P. W., CURTIS, P. J., AND HEMMING,H. G. 1946 A substance causing abnor-mal development of fungal hyphae pro-duced by Penicillium janczewskii Zal. I.Biological assay, production, and isolationof "curling factor." Brit. Mycol. Soc.Trans., 29, 173-187.

16. BURTON, H. S. 1955 Antibiotics fromStreptomycetes. The actiduins. Chem. &Ind., 1955, 442-443.

17. CASTLE, H. AND KUBSCH, F. 1949 Theproduction of usnic, didymic and rhodo-caldonic acids by the fungal component ofthe lichen Caldonia cristatella. Arch. Bio-chem., 23, 158-160.

18. CELMER, W. D., MURAI, K., RAO, K. V.,TANNER, F. W., JR., AND MARSH, W. S.1957 The quinocycline complex. I. Isola-tion and characterization. AntibioticsAnn., 1957/1958, 484-492.

19. Chas. Pfizer and Co., Inc. 1959 Fermenta-tion of a tetracycline-producing Strepto-myces. British Patent Spec. 817,730.

20. CHUN, D. 1956 Distribution of the anti-biotic-producing Streptomyces in the south-western area of the Republic of Korea.Antibiotics & Chemotherapy, 6, 324-329.

21. CLUTTERBUCK, P. W., MUKHOPADHYAY, S. L.,

OXFORD, A. E., AND RAISTRICK, H. 1940Studies in the biochemistry of microorgan-isms.65 (A) A survey of chlorine metabolismby moulds; (B) Caldariomycin, C5HO2Cl2,a metabolic product of Caldaricmycesfumago Wor. Biochem. J., 34, 664-677.

22. CURD, F. H., ROBERTSON, A., AND STEPHEN-SON, R. J. 1933 Lichen acids. IV. Atra-norin. J. Chem. Soc., 1933, 130-133.

23. CURTIN, T. P. AND REILLY, J. 1940 Sclero-tiorine, C2oH2105Cl, a chlorine-containingmetabolic product of Penicilliumn sclero-tiorum Van Beyma. Biochem. J., 34, 1419-1421.

24. DEAN, F. M., EADE, R. A., MOUBASHER, R.A., AND ROBERTSON, A. 1957 Fulvicacid, its structure and relationship tocitromycetin and fusarubin. Nature, 179,366.

25. DEAN, F. M., ERNI, A. D. T., ANDROBERTSON, A. 1956 The chemistry offungi. XXVJ. Dechloronornidulin. J.Chem. Soc., 1956, 3545-3548.

26. DEAN, F. M., ROBERTS, J. C., ANDROBERTSON, A. 1954 The chemistry offungi. XXII. Nidulin and nornidulin("ustin"), chlorine-containing metabolicproducts of Aspergillus nidulans. J.Chem. Soc., 1954, 1432-1439.

27. DEAN, F. M., ROBERTSON, A., ROBERTS, J.C., AND RAPER, K. B. 1953 Nidulin and"ustin", two chlorine-containing meta-bolic products of Aspergillus nidulans.Nature, 172, 344.

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